H5 Serotype Influenza Viruses
Influenza viruses (IV) belong to the Orthomyxoviridae family, which currently contains six different genera (ICTV 2014). Among them, Influenzavirus A genus is of the highest eidemiological importance. Influenza virus type A is surrounded by a protein-lipid envelope and contains genome in the form of single-stranded ribonucleic acid (RNA) with negative polarity, which is divided into eight segments and codes for 10 proteins.
Type A IV strains are classified according to the serotype of surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Up till now, 18 HA serotypes and 11 NA serotypes (Szewczyk B. et al., 2014) have been identified. HA and NA proteins play an important role in virus replication cycle. By binding to sialic acid residues on host cell receptors, HA enables IV internalisation by endocytosis and then it mediates the fusion of viral envelope with cell membrane, which leads to a release of virus's genetic material into the cytoplasm and initiates process of its replication (Skehel J. J. and Wiley D. C., 2000). NA catalyses cleavage of glycoside bonds with sialic acid, which conditions release of the virus from attacked cells and therefore its spreading (Lamb R. A. and Choppin P. W., 1983).
Surface glycoproteins of IVs undergo gradual changes as a result of RNA polymerase errors and selection pressure from host immunological system. This phenomenon, referred to as antigen drift, leads to the escape of the virus out of immunological system control and usually causes limited illnesses, rarely epidemics. Greater and more rapid changes of viral antigens, referred to as antigen shift, are a result of genetic reassortation and can lead to pandemia. Variation makes IVs a real challenge for influenza diagnostics, prevention and treatment.
In birds, there are influenza viruses (AIVs) being a combination of 16 HA serotypes and 9 NA serotypes. There are 2 main type A IV serotypes are circulating in human population: H1N1 and H3N2. Influenza viruses are host-specific, so that AIVs usually do not infect humans. Among numerous AI viruses, low-pathogenic (LP) strains are dominant, causing mild infections. H5 and H7 serotypes influenza viruses, usually non-pathogenic in their natural hosts, which are wild birds, especially water birds, may become highly-pathogenic (HP) after transfer to an infection-prone population of domestic birds and by their intermediary become a risk for the health and life of humans. That was the case for HPAIV H5N1 strain.
Since the disease outbreak in 1996 at the goose-farm and human infections in 1997 in China (Xu X. et al., 1999), spreading of HPAI H5N1 viruses has been observed (Verhagen J. H. et al. 2015). The disease outbreaks among birds, often reaching epizootic scale, cause high animal mortality and force stamping out domestic birds. Cases of fatal infection of humans with H5N1 viruses are still reported. The risk of reassortation of circulating H5N1 AIVs with mammal viruses, leading to new strains capable of direct transmission between humans, is remaining.
During circulation and spreading of H5N1 viruses, HA genes were differentiating to the large number of genetic lines, referred to as clades (Verhagen J. H. et al., 2015). From 2009 onward, the emergence of the reassortants of H5-subtype HPAIVs, such as H5N2, H5N5, H5N6 and H5N8, has been noted. After their identification in 2014, H5N8 HPAIs are observed to spread rapidly in domestic birds population. As a result of reassortation with HPAIV H5N8, novel H5N2 HPIV emerged and was identified at the disease outbreaks at the chicken and turkey farms in Canada at the end of 2014 (Ip H. S. et al., 2015). In 2015, in the USA, H5N2 HPAIV caused high poultry mortality (Hvistendahl M., 2015).
H5 Hemagglutinin—Target Antigen for Monoclonal Antibodies
HA is synthesized as a single polypeptide chain, referred to as a HA precursor—HA0, containing signal sequence, HA1 and HA2 subunits separated by proteolytic enzyme cleavage site. HA0 cleavage is necessary to HA activation conditioning IV infectiveness, and protein susceptibility to digestion by proteolytic enzymes is one of the factors determining viral pathogenicity (Steinhauer D. A., 1999). In H5 HA proteins, cleavage site contains multiple basic amino acids, which makes the protein susceptible to the digestion by proteases active in most tissues, and the infection with H5 serotype IV has systemic nature.
The studies of structure of HA proteins of various serotypes, including H5, showed that antigenically-different hemagglutinins are structurally similar and show the same sub-domain organization (Ha Y. et al., 2002, Wilson I. A. Et al., 1981). The protein is composed of globular domain formed by HA1 subunit and stem domain formed mainly by HA2 subunit and N- and C-terminal amino acid sequences of HA1 subunit. Globular domain of HA contains antigen binding domain (RBD, through which virus binds host cell, whereas stem domain mediates fusion of viral and endosomal membrane. HA2 subunit within stem domain is responsible for HA trimer formation and stabilization, as well as its anchoring within viral lipid envelope, where the protein forms characteristic structures.
HA is the most variable IV antigen. This applies, in particular, to the globular domain. Mutation-susceptible protein domain contains epitopes for neutralizing antibodies usually having limited range of specificities within the serotype. In case of H5N1 HPAIV, weak cross-reactivity of those antibodies against antigenically distant viruses from different clades is observed. Antibodies directed against globular domain neutralize infectiveness of the viruses mainly by interfering with their binding to host cell receptors. Activity of RBD-binding neutralizing antibodies is observed in vitro as an inhibition of IV hemagglutinin activity.
In contrary to the globular domain-forming HA1 subunit, HA2 subunit, which forms an essential part of stem region, is relatively well conserved and contains epitopes for neutralizing antibodies of broad specificity range against different viral serotypes. Antibodies directed against stem domain neutralize viral infectiveness by blocking fusion of the membranes.
Use of Monoclonal Antibodies in Immunoprophylaxis and Immunotherapy
Currently, infections evoked by HPAIVs are controlled by using antiviral drugs and active or passive immunization. Immunoprophylaxis and immunotherapy are preferred due to the observed increase of IVs drug-resistance and side effects of pharmacotherapy. Preventive actions comprise mainly preventive vaccinations. Traditional vaccine production methods are long-lasting and would not be able to generate sufficient number of doses in case of pandemic risk. Immunization with the use of those vaccines is often associated with undesirable side effects. Alternative for conventional vaccines are DNA vaccines and subunit vaccines based on recombinant HA proteins. H5 HA proteins are produced mainly in eukaryotic cells, but also in bacterial cells. The main challenge for work on the development of subunit vaccines against influenza is the production of antigen having characteristics of native HA. Particularly important for vaccine HA quality is correctness of structure of HA1 subunit, where conformational epitopes for neutralizing antibodies are localized and protein oligomerization.
Anti-HA monoclonal antibodies (mAbs) can be use during the development of influenza vaccines and at various stages of their production and implementation. The first indicator of usefulness of HA proteins for the vaccine production are the results of the antigenicity tests carried out with the use of well-characterized antibodies (Chiu F. F. et al., 2009). Particularly useful in this regard are mAbs recognizing conformational, neutralizing HA epitopes. mAbs can also be used in studies of the level of HA proteins oligomerization, beside erythrocytes agglutination and fetuin binding tests. Using mAbs in affinity chromatography or immunoprecipitation, one can also isolate and purify H5 HA proteins. Antigens isolated and purified in this way can be used as components of prototype vaccines against IVs of H5 serotype, intended for initial evaluation of vaccine ability to evoke protective immunological response in animals. mAbs can also be used in the control of H5 HA vaccine stability, storaged separately or in immunogenic compositions. The most important use of serotype-specific mAbs associated with active immunisation is their use in so-called DIVA tests (Differentiation of Infected From Vaccinated Animals). Providing possibility to differentiate infected and vaccinated animals is the principal requirement for carrying out vaccinations against avian influenza (Suarez D. L., 2005).
Another way of preventing IV infections, but mostly of influenza treatment, is the use of mAbs for passive immunization. Those are mainly humanized antibodies or, preferably, antibodies produced de novo, as human mAbs. Predictably, neutralizing antibodies directed against variable and mutation-prone HA1 subunit of H5 hemagglutinin may not be effective against heterologous viral strains or antigen-drifted strains. In this context, it is of great importance to obtain neutralizing mAbs against epitops of HA1 subunit of HA, which are conserved among H5N1 IVs (Cao Z. et al., 2012, Du L. et al., 2013, Oh H. L. Et al., 2010, Wu R. et al., 2014). In the recent years, there are increasing reports about generating mAbs recognizing conservative epitopes in HA2 subunit of HA stem region, which show heteroserotypic neutralizing activity (Corti D. et al., 2011, Ekiert D. C. Et al., 2009, Okuno Y. et al., 1993, Sui J. et al., 2009).